Dear all,

I was wondering if I could get advice on a complicated model for DESeq2? Currently, I have different mutations in two cell lines that are tissue-relevant (neurons and glia) with multiple replicates for each. Essentially, I have a double knockout (0), heterozygous knockout (1), wild type (2), and duplication (4) for each of these mutations (gene dosage). I am trying to make a model in which I can compare to wildtype and account for the different cell lines and or mutations. I'm not sure whether I should separate the cell lines and analyze seperately, and then look at overlap. Or if I should include the cell line into the model. I've listed my variables below. Condition is wildtype or mutant.

Both GENE1 and GENE2 are relevant for the same disease.

Celltype condition dosage RIN MutationGene

1 WT 2 9 GENE1
1 WT 2 9 GENE1
1 WT 2 8.2 GENE1

1 Mutant 1 7 GENE1
1 Mutant 1 9 GENE1
1 Mutant 1 8.2 GENE1

1 Mutant 0 7 GENE1
1 Mutant 0 10 GENE1
1 Mutant 0 9.2 GENE1

1 Mutant 3 9.2 GENE1
1 Mutant 3 8.3 GENE1
1 Mutant 3 9 GENE1

2 WT 2 9 GENE1
2 WT 2 9 GENE1
2 WT 2 8.2 GENE1

2 Mutant 1 7 GENE1 
2 Mutant 1 9 GENE1
2 Mutant 1 8.2 GENE1

2 Mutant 0 7 GENE1
2 Mutant 0 10 GENE1
2 Mutant 0 9.2 GENE1

2 Mutant 3 9.2 GENE1
2 Mutant 3 8.3 GENE1
2 Mutant 3 9 GENE1

3 WT 2 9 GENE2
3 WT 2 9 GENE2
3 WT 2 8.2 GENE2

3 Mutant 1 7 GENE2
3 Mutant 1 9 GENE2
3 Mutant 1 8.2 GENE2

3 Mutant 0 7 GENE2
3 Mutant 0 10 GENE2
3 Mutant 0 9.2 GENE2

3 Mutant 3 9.2 GENE2
3 Mutant 3 8.3 GENE2
3 Mutant 3 9 GENE2

4 WT 2 9 GENE2
4 WT 2 9 GENE2
4 WT 2 8.2 GENE2

4 Mutant 1 7 GENE2
4 Mutant 1 9 GENE2
4 Mutant 1 8.2 GENE2

4 Mutant 0 7 GENE2
4 Mutant 0 10 GENE2
4 Mutant 0 9.2 GENE2

4 Mutant 3 9.2 GENE2
4 Mutant 3 8.3 GENE2
4 Mutant 3 9 GENE2

My two main questions are:

#1) Should I separate them by gene and/or cell line? The cell lines are related (i.e. neuron and glia)
#2) Does this model make sense? 

~condition + condition:dosage + condition:Celltype + RIN + dosage:MutationGene? I get an error unfortunately.   

# Ive also tried
~dosage + dosage:Celltype + RIN + dosage:MutationGene

Apologies for the complexity.

Thanks a lot!

A few thoughts;

1. The only degree of relatedness between these two cell types is that they happen to be physically close to each other in vivo (like, in the brain), however their expression profiles, function, etc. between neurons and glia (microglia? astrocyttes? oligodendrocytes?) are about as different as can be.

2. Your model is likely a lot more complex than it needs to be.

What is the actual question you are trying to answer? Do you want to see if the same genetic perturbation has a similar transcriptional effect on the different cell types? I'd start a bit more modestly: simply comparing the logFC of the KO vs WT in the neuron line vs the same logFC in the glial line might be a good place to start ...